1. Field of the Invention
The present invention relates to purification methods, and more particularly, to a fusion protein comprising a target protein and a protease prodomain protein wherein the prodomain protein has high affinity for binding with a corresponding protease or variant thereof to provide a protease binding complex for subsequent recovery of the target protein.
2. Description of Related Art
Recombinant DNA techniques have facilitated the expression of proteins for diverse applications in medicine and biotechnology. However, the purification of recombinant proteins is often complicated and problematic. The large-scale, economic purification of proteins generally includes producing proteins by cell culture, such as bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.
Once a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different techniques. As part of the overall recovery process for the protein, the protein may be exposed to an immobilized reagent, which binds to the protein.
Proteomics initiatives of the post genomic era have greatly increased the demand for rapid, effective and standardized procedures for the purification and analysis of proteins. For example, recombinant proteins are frequently fused with other proteins or peptides to facilitate purification. The fused domain serves as a temporary hook for affinity purification and ultimately must be cleaved off by site-specific proteolysis. A number of fusion protein systems using different carrier proteins are now commercially available, particularly for E. coli expression. Examples include maltose binding protein, glutathione S-transferase, biotin carboxyl carrier protein, thioredoxin and cellulose binding domain.
Fusion protein expression simplifies the separation of recombinant protein from cell extracts by affinity chromatography using an immobilized, moderate-affinity ligand specific to the carrier protein. However, typically, immobilization requires the covalent attachment of the ligand to the matrix resulting, in many cases, in loss of activity. A typical example of a widely used product is Protein A-Sepharose. This highly expensive product is used for the purification of IgG by affinity chromatography, as well as for many diagnostic protocols.
Thus, more economical and technically simple methods for purification of soluble proteins, which do not involve scale-up of chromatographic procedures, are therefore desirable.
The function of proteases range from broad specificity, degradative enzymes to highly sequence specific enzymes that regulate physiological processes from embryonic development to cell death. Some high specificity proteases have been recruited from nature to serve as tools for the purification and analysis of proteins in a manner somewhat analogous to use of restriction endonucleases to manipulate DNA. The specific processing enzymes currently available are from mammalian sources, such as thrombin, factor Xa and Enteropeptidase. However, although widely used in protein work these natural enzymes are very expensive and of low stability limiting their usefulness for many applications.
Considerable effort has been devoted to engineering robust, bacterial proteases, such as subtilisin, to cleave defined sequences. Subtilisin is a serine protease produced by Gram-positive bacteria or by fungi. Subtilisins are important industrial enzymes as well as models for understanding the enormous rate enhancements affected by enzymes. The amino acid sequences of numerous subtilisins are known and include subtilisins from Bacillus strains, for example, subtilisin BPN′, subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesenticopeptidase. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980's. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. The most mutagenized subtilisins (Neet K E, et al., Proc Natl Acad Sci USA 1966, 56:1606-1611; Polgar L, et al., Biochemistry 1967, 6:610-620) are those secreted from the Bacillus species amyloliquefaciens (BPN′), subtilis (subtilisin E) and lentus (Savinase).
In spite of the intense activity in protein engineering of subtilisin it previously has not been possible to transform it from a protease with broad substrate preferences into an enzyme suitable for processing specific substrates thereby rendering it useful for protein recovery systems. Thus, it would be extremely useful for research and protein purification to be able to use low specificity proteases such as subtilisin for purification processes.